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• 批准号: 10182761
• 负责人: Michael Ailion
• 金额: $ 20万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10182761
• 关键词: Attention deficit hyperactivity disorder; Beta Cell; Binding; Biochemical; Biogenesis; Biogenic Amines; Biological; Biological Assay; Biological Process; Blood Glucose; Caenorhabditis elegans; Calcium; Cell Line; Cells; Chemicals; Complex; Defect; Dense Core Vesicle; Development; Diabetes Mellitus; Disease; Eating Disorders; Endocrine; Equipment; Genetic; Genetic Screening; Goals; Golgi Apparatus; Growth Factor; Hormone secretion; Hormones; Human; In Vitro; Lead; Mediating; Membrane; Mental Depression; Mental disorders; Metabolic Diseases; Molecular; Monomeric GTP-Binding Proteins; Mood Disorders; Nematoda; Nerve; Neuroendocrine Cell; Neurons; Neuropeptides; Obesity; Organelles; Process; Proteins; Role; Schizophrenia; Sorting - Cell Movement; Synaptic Vesicles; Synaptic plasticity; Testing; Work; blood glucose regulation; golgin; in vivo; insight; mutant; neuron development; neuronal survival; novel therapeutics; pain sensation; peptide hormone; rab GTP-Binding Proteins; response; trafficking; trans-Golgi Network; vesicular release

Project summary The dense-core vesicle is an organelle that releases peptide hormones, growth factors, and biogenic amines from neurons and neuroendocrine cells in response to increases in calcium. Dense-core vesicle cargos regulate a variety of biological processes including neuronal survival and development, pain sensation, blood glucose homeostasis, and synaptic plasticity. As a consequence, numerous diseases such as mood disorders, obesity, and diabetes are caused by defects in neuropeptide and hormone secretion. Thus, it is important to determine the molecular machinery and mechanisms that orchestrate the biogenesis and release of these vesicular carriers. However, in comparison to other vesicular compartments such as synaptic vesicles, little is known about the molecular mechanisms of dense-core vesicle biogenesis, trafficking, and release. Dense-core vesicles are generated at the trans-Golgi and gain their compartmental identity in a poorly defined maturation process that occurs post-Golgi, but few molecules have been identified that function in these processes. Cargo sorting to dense-core vesicles remains a puzzle. The long-term goal of this project is to understand the molecular mechanisms by which dense-core vesicles are formed, sort cargos, and gain their compartmental identity. A first step towards this goal is to identify molecules required for dense-core vesicle biogenesis. We performed a genetic screen in the nematode C. elegans for mutants defective in dense-core vesicle function and identified a number of new proteins that act in dense-core vesicle biogenesis, including the small GTPase RAB-2 and CCCP-1, a RAB-2 effector, as well as the EARP endosomal trafficking complex. In this project, we will test how RAB-2 and its effectors interact with EARP to mediate cargo sorting to dense-core vesicles. In Aims 1 and 2, we will use genetic, biochemical and cell biological approaches in C. elegans and in the mammalian insulin-secreting cell line INS-1 832/13 to determine how and where the RAB-2 and EARP complexes are localized, whether they physically and functionally interact, and determine their precise roles in the sorting, processing, and secretion of dense-core vesicle cargos. In Aim 3, we will use a combination of in vitro biochemical approaches and in vivo functional assays to determine how the golgin-like coiled-coil protein CCCP-1 binds membranes and functions to mediate dense-core vesicle biogenesis. These studies will advance our understanding of how dense-core vesicles are generated and sort cargos, and provide general insights into mechanisms of membrane trafficking and cargo sorting controlled by multisubunit complexes that mediate trafficking between endosomal compartments and the trans-Golgi network.

项目总结 致密核小泡是释放多肽激素、生长因子和生物胺的细胞器。 神经元和神经内分泌细胞对钙含量增加的反应。密核泡囊货物 调节多种生物过程，包括神经元的生存和发育、痛觉、血液 葡萄糖动态平衡和突触可塑性。其结果是，许多疾病，如情绪障碍， 肥胖和糖尿病是由神经肽和激素分泌缺陷引起的。因此，重要的是要 确定协调这些物质的生物发生和释放的分子机制和机制 泡状载体。然而，与其他囊泡相比，如突触小泡，几乎没有 了解致密核小泡生物发生、运输和释放的分子机制。致密核 囊泡是在跨高尔基体产生的，并在一个不明确的成熟过程中获得了它们的房室身份 发生在高尔基体后的过程，但很少有分子被确定在这些过程中起作用。货货 对致密核小泡的分类仍然是一个谜。该项目的长期目标是了解 致密核小泡形成的分子机制，对货物进行分类，并获得它们的隔间 身份。迈向这一目标的第一步是确定致密核心囊泡生物发生所需的分子。我们 对线虫进行致密核小泡功能缺陷突变体的遗传筛选 并确定了一些在致密核心囊泡生物发生中起作用的新蛋白质，包括小GTP酶 RAB-2和CCCP-1，RAB-2效应器，以及EARP内体运输复合体。在这个项目中，我们 将测试RAB-2及其效应器如何与EARP相互作用，将货物分拣到致密核小泡。在……里面 目标1和目标2，我们将在线虫中使用遗传、生化和细胞生物学方法 哺乳动物胰岛素分泌细胞系INS-1 832/13确定RAB-2和EARP的途径和位置 复合体是本地化的，无论它们在物理和功能上相互作用，并确定它们在 分拣、加工和分泌致密核小泡的货物在目标3中，我们将使用中的组合 体外生化方法和体内功能分析来确定高尔金样卷曲蛋白 CCCP-1结合细胞膜和功能，介导致密核小泡的生物发生。这些研究将 促进我们对致密核心小泡是如何产生和分类货物的理解，并提供一般 由多亚单位复合体控制的膜运输和货物分拣机制的洞察 调解内体隔室和跨高尔基网络之间的运输。